Derivation and culture of putative parthenogenetic embryonic stem cells in new gelatin substrates modified with galactomannan

Abstract

Human embryonic stem cells (ESC) lines to be used for cell therapies must be created and maintained under strict conditions, excluding the use of undefined supplements. Two key steps in the creation of a new embryonic stem cell line are adherence to the substrate and derivation towards the formation of a primary colony. The bovine parthenote embryo model was used to test different matrices of gelatin nanofibers and gelatin/galactomannan films to be used for ESC derivation and culturing. Gelatin/galactomannan films were made in two concentrations of galactomannan, 0.1 and 0.3%, in an aqueous solution of gelatin and tested for gel cytotoxicity using cumulus cells (CCs). CCs showed normal cell morphology, with no sign of lysis or degeneration in any of the matrices tested. Inner cell masses of parthenote blastocysts (n=116) were placed onto the gel matrices for culture. There were three or four repeats for each matrix. Our results showed a good rate of inner cell mass (ICM) adherence on the gelatin/galactomannan films (41%–44%) and one derivative of the gel nanofiber (17% adherence to the substrate). These results encouraged us to try new gelatin formulations to increase the rates of derivation and cell proliferation under defined culture conditions to comply with good manufacturing practice directives for the potential therapeutic use of ESCs.